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SKIN CORROSION TESTUSING THE RECONSTRUCTED HUMAN MODEL“LABCYTE EPI-MODEL24”
Ver. 1.6

LabCyte EPI-MODEL24 SKIN CORROSION TEST

S.O.P.

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TABLE OF CONTENTS
1.

RATIONALE AND BACKGROUND ............................................................................................... 4
1.1
1.2
1.3

LabCyte EPI-MODEL24 SKIN CORROSION TEST (LabCyte EPI-MODEL24 SCT) ............. 4
BACKGROUND OF THE LabCyte EPI-MODEL24 SCT ......................................................... 4
BASIS OF THE METHOD ....................................................................................................... 4

1.4
1.5
1.6

LIMITATION OF THE METHOD .............................................................................................. 6
BRIEF BASIC PROCEDURE .................................................................................................. 6
DATA INTERPRETATION PROCEDURE (PREDICTION MODEL) ........................................ 6

1.3.1
1.3.2

2.

MATERIALS ................................................................................................................................... 8
2.1

LabCyte EPI-MODEL24 .......................................................................................................... 8

2.2
2.3
2.4

TEST CHEMICALS ................................................................................................................. 8
CONSUMABLES ..................................................................................................................... 8
OTHERS.................................................................................................................................. 9

2.1.1
2.1.2
2.1.3

2.4.1
2.4.2

3.

EQUIPMENT / INSTRUMENTS ........................................................................................................ 9
CONSUMMABLE ITEMS .................................................................................................................. 9

TEST METHOD ............................................................................................................................ 10
PREPARATIONS ................................................................................................................... 10

3.2

TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT ........... 11

3.3

EXECUTION OF THE TEST ................................................................................................. 14

3.2.1
3.2.2
3.3.1
3.3.2
3.3.3
3.3.4

NEGATIVE CONTROL SUBSTANCE ........................................................................................... 10
POSITIVE CONTROL SUBSTANCE............................................................................................... 10
POLY WASH BOTTLE FOR PBS.................................................................................................... 10
MTT SOLUTION ............................................................................................................................. 10
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE .....................................................11
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT ................................................... 12
PREPARATION OF LabCyte EPI-MODEL24 (DAY -1) ................................................................... 14
APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0) ..................................................... 14
MTT ASSAY (DAY 0) ....................................................................................................................... 18
FORMAZAN EXTRACTION AND MEASUREMENT (DAY 0-1) ...................................................... 18

ASSESSMENT ............................................................................................................................. 20
4.1
4.2

5.

LabCyte EPI-MODEL24 KIT COMPONENTS ................................................................................... 8
SHIPMENT OF LabCyte EPI-MODEL24 ........................................................................................... 8
INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL24 ................................................................. 8

3.1

3.1.1
3.1.2
3.1.3
3.1.4

4.

TEST SYSTEM DESCRIPTION ........................................................................................................ 5
ASSAY QUALITY CONTROL ............................................................................................................ 5

CONDITIONS FOR A SUCCESSFUL STUDY ...................................................................... 20
ASSAY CRITERIA ................................................................................................................. 20

REFFERENCE .............................................................................................................................. 21

MDS 1-1:

RECEIPT OF LABCYTE EPI-MODEL24 .......................................................................... 22

MDS 1-2: MTT REACTION INTERFERENCE TEST (DETECTION OF THE CHEMICALS THATSTAIN THE TISSUE) STEP-1 (Section 3.2.1.1) ................................................................................. 23
MDS 1-3: MTT REACTION INTERFERENCE TEST (DETECTION OF CHEMICALS THATDIRECTLY REDUCE MTT) STEP-3 (Section 3.2.2.1) ........................................................................ 24
MDS 1-4:

PREPARATION OF FREEZE-KILLED TISSUE (Section 3.2.2.2)................................... 25

MDS 2:

PRE-INCUBATION OF LABCYTE EPI-MODEL24 (Section 3.3.1) .................................... 26

MDS 3-1:

APPLICATION OF TEST CHEMICALS AND RINSING: 3 MINUTES EXPOSURE (Section

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3.3.2)..................................................................................................................................................... 27
MDS 3-2: APPLICATION OF TEST CHEMICALS AND RINSING: 60 MINUTES EXPOSURE(Section 3.3.2) ..................................................................................................................................... 29
MDS 4:MTT ASSAY (Section 3.3.3) ................................................................................................ 31

MDS 5:FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.4) ................................. 32

REVISION HISTORY ............................................................................................................................ 33

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1. RATIONALE AND BACKGROUND
1.1 LabCyte EPI-MODEL24 SKIN CORROSION TEST (LabCyte EPI-MODEL24 SCT)
The LabCyte EPI-MODEL24 skin corrosion test (LabCyte EPI-MODEL24 SCT) is designed forthe prediction of acute skin corrosion of chemicals by measurement of their cytotoxic effect, as
reflected in the MTT assay, on the Reconstructed Human Epidermis (RhE) model. LabCyte
EPI-MODEL24 tissues are commercially available with a minimum of 24 tissues (one 24-wellplate) per order.

1.2 BACKGROUND OF THE LabCyte EPI-MODEL24 SCT
Skin corrosion refers to the production of irreversible tissue damage in the skin following theapplication of a test chemical, as defined by the Globally Harmonized System (GHS) for
Classification and Labeling of Chemical Substances and Mixtures1).
The potential for chemical induced skin corrosion is an important consideration in establishingprocedures for the safe handling, packaging and transport of chemicals. Various systems forclassification of corrosive potential are included in international regulatory requirements.
The present test is based on the fact that corrosive chemicals are cytotoxic after a short term
exposure to the stratum corneum of the epidermis, if cytotoxicity is immediately determined after
chemical exposure. It is designed to predict and classify the skin corrosive potential of a chemical
by using a three dimensional human epidermis model.
In 1998, EPISKIN and TER in vitro corrosive tests were successfully validated and met theacceptance criteria previously defined by the Management Team of the ECVAM International
Validation Study2). Because EPISKIN was not available after the study, a catch up validation study
was performed with the RhE model, EpiDerm3).
In 2002, national coordinators of the OECD Test Guideline Programme (WNT) endorsed New
Draft Test Guidelines (TG) 430 (TER) and 431 (Human Skin Model; adopted in 13th April 2004) for
In Vitro Skin Corrosion Testing. In the OECD TG431, general functional and performance criteria
were defined in case other (or new) skin or epidermis models were to be used in the context of thisguideline4). The current SOP describes a generally applicable method for Skin Corrosion Testing,here applied to the LabCyte EPI-MODEL24 SCT.

1.3 BASIS OF THE METHOD
Most international regulatory classification schemes define chemically induced dermal corrosion
as full thickness destruction (necrosis) of the skin tissue, while some extend the definition of
corrosion to include any irreversible alterations caused to the skin. The potential to induce skincorrosion is an important consideration in establishing procedures for the safe handling,
packaging and transport of chemicals.
The test is designed to predict and classify the skin corrosive potential of a test substance byassessment of its effect on a reconstructed human epidermis. The relative viability of the treatedtissues is measured at the end of the treatment exposure (3 minutes and 60 minutes), by the MTT[(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide] assay. The cell viability of thenegative control is then considered and used to classify test substances. The culture environment
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might allow the detection of very small quantities of cytokines secreted by the epidermis in
response to topical application of test substances.

1.3.1
TEST SYSTEM DESCRIPTION
LabCyte EPI-MODEL24 is a commercially available RhE model produced by Japan Tissue
Engineering Co., Ltd. It consists of normal human epidermal keratinocytes whose biologicalorigin is neonate foreskin. In order to maintain their phenotype during expansion, humankeratinocytes are cultured on a feeder layer of 3T3-J2 cells (Rheinwald and Green, 1975; Green,1978). Reconstruction of human cultured epidermis is achieved by cultivating and proliferating
keratinocytes on an inert filter substrate (surface 0.3 cm2) at the air-liquid interface for 13 days with
an optimized medium containing 5% fetal bovine serum. The model reconstructs a multilayer
structure consisting of a fully differentiated epithelium with features of the normal human epidermis,
including a stratum corneum. LabCyte EPI-MODEL24 is embedded in an agarose gel containing
nutrient solution and shipped in 24-well plates.
1.3.1.1 QUALITY CONTROL OF THE TEST SYSTEM
The LabCyte EPI-MODEL24 is manufactured according to defined quality assurance
procedures. Each product is provided with directions concerning storage conditions, instructions
for use, lot number and origin. The quality of each batch production is assessed by histologicalexamination (demonstration of human epidermis-like structure with multilayered stratum corneum),
MTT assay to determine cell viability, and by IC50 with SLS (sodium lauryl sulfate) to analyze
barrier function integrity (0.14 ≤ IC50 ≤ 0.4).

1.3.1.2 PRECAUTIONSThe epidermal cells are taken from a healthy donor negative to HIV and Hepatitis. Nevertheless,handling procedures for biological materials should be followed:a) It is recommended to wear gloves during handling of the model and kit components.
b) After use, the epidermis, as well as the material and all media that were in contact with it,
should be decontaminated prior disposal (e.g. using special containers or autoclaving).

1.3.2

ASSAY QUALITY CONTROL

1.3.2.1 ASSAY ACCEPTANCE CRITERION 1: NEGATIVE CONTROLThe absolute OD of the negative control tissues (NC, treated with sterile distilled water) in the
MTT assay is an indicator of cell viability obtained in the testing laboratory after shipping andstoring procedures and under specific conditions of use.
0.7 ≤ Mean OD (A570/650) measured value ≤ 2.5

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1.3.2.2 ASSAY ACCEPTANCE CRITERION 2: POSITIVE CONTROL
8N (8 mol/L) potassium hydroxide (KOH) solution is used as positive control (PC) and tested
concurrently with the test chemicals. Here, concurrent means that the PC has to be tested in eachassay, but not more than one PC is required per testing day.
Mean cell viability (60 minutes) < 15%
1.3.2.3 ASSAY ACCEPTANCE CRITERION 3: DIFFERENCE OF VIABILITY
Since skin corrosion potential is predicted in each test from the mean viability determined on 2
single tissues, the variability of tissue replicates should be acceptably low.
In the viability range of 20-100%, and for ODs≥ 0.3, difference of viability between the
two tissue replicates should not exceed 30%.

1.4 LIMITATION OF THE METHOD
One limitation of this assay method is a possible interference of the test substance with the MTT
endpoint. A colored test substance or one that directly reduces MTT (and thereby mimics
dehydrogenase activity of the cellular mitochondria) may interfere with the MTT endpoint. However,these test substances are a problem only if at the time of the MTT test (i.e. after test substance
exposure) sufficient amounts of the test substance are still present on (or in) the tissues. In case ofthis unlikely event, the (true) metabolic MTT reduction and the contribution by a colored test
material or (false) direct MTT reduction by the test material can be quantified by a proceduredescribed in Section 3.2.
The method is not designed for testing of highly volatile test substances, gases and aerosols.

1.5 BRIEF BASIC PROCEDURE
On the day of receipt, LabCyte EPI-MODEL24 tissues are transferred to fresh assay medium
and conditioned by incubation to release transport-stress related compounds and debris
overnight. After overnight pre-incubation, LabCyte EPI-MODEL24 tissues are topically exposedto the test chemicals for 3 minutes and 60 minutes. Two tissues each are used per treatment,
negative control and positive control. After exposure, tissues are rinsed and blotted, and assay
medium is replaced by MTT-assay medium. After 3 hours incubation, the blue formazan salt is
extracted with isopropanol. The optical density of the formazan extract is determined
spectrophotometrically at 570 nm and 650 nm as reference, and cell viability is calculated foreach tissue as % of the mean of the negative control tissues. Skin corrosive potential of the test
materials is classified according to the remaining cell viability obtained after 3 minutes or 60
minutes exposure with the test chemical.

1.6 DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)
Corrosive potential of the test materials is predicted from the relative mean tissue viabilities
obtained after 3 minutes as well as 60 minutes treatment compared to the negative control tissues
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concurrently treated with H2O. The prediction models for the LabCyte EPI-MODEL24 SCT
methods, associated with the UN GHS classification system, are shown in Table 1.
Table 1. LabCyte EPI-MODEL24 SCT prediction model
Step 13 minutes
60 minutes
Prediction< 50%
Corrosive
Viability
< 15%
≥ 50%
≥ 15%
Non-corrosive

Step 23 minutes
< 15%
≥ 15%

Prediction
Sub-category 1A
Sub-category 1Band 1C7

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2. MATERIALS
2.1

LabCyte EPI-MODEL24

2.1.1
LabCyte EPI-MODEL24 KIT COMPONENTS
LabCyte EPI-MODEL24 kit components are shown in Table 2.
Table 2. LabCyte EPI-MODEL24 Kit Components
Component

Qty

LabCyte EPI-MODEL24 plate

Description
Contains 24 culture inserts with tissues fixed in

1 platenutritive agar medium for transport (usable area:2

0.3 cm ).
Assay Medium
24-well plate

2.1.2

1 bottle
1 plateBasic medium for incubation (30 mL). Store at
refrigeration temperature.
Blank

plate

for

use

in

assay.

Storeatroom-temperature.

SHIPMENT OF LabCyte EPI-MODEL24

LabCyte EPI-MODEL24 is packed in a special container (Icompo/NIPPON EXPRESS CO.,LTD) and delivered by NIPPON EXPRESS CO., LTD. After the Icompo is delivered, examine thecontents and make sure that all kit components (LabCyte EPI-MODEL24 plate, assay medium,and 24-well assay plate) are included in the package. Verify lot number and expiration date, andrecord details in the Methods Documentation Sheet (MDS) 1.
NIPPON EXPRESS will pick up the Icompo at a later date (generally, the day after the date ofdelivery), and we ask that you return it with the insulating materials.

2.1.3

INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL24

Start incubating all culture inserts soon after opening the package. Do not store the culture
inserts in the nutritive agar after opening the aluminum package.
The human epidermis cells used in LabCyte EPI-MODEL24 originate from a healthy donor andare HIV-, HBV-, HCV-, and HPV-negative. However, handle them with enough care and inaccordance with the laboratory biosafety guidelines since they contain raw materials of humanorigin.

2.2 TEST CHEMICALS
Coded test chemicals are delivered to each laboratory.

2.3

CONSUMABLES

The following consumables are required.
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* The described quantities are necessary so that 1 to 2 samples can be assayed once.
• Assay Medium, 100 mL (J-TEC: 402100)
1 bottle• MTT (e.g. J-TEC: 403026)
1 bottle• 24-well assay plate (Corning: 353047)
3 plates• 96-well plate (e.g. Corning: 353072)
1 plates• Phosphate buffered saline (PBS) 500 mL (e.g. Invitrogen: 14190-144)
3 bottles• Isopropanol 500 mL (e.g. Wako Pure Chemical Industries: 164-08335)
1 bottle• 8 mol/l KOH 500 mL (e.g. Wako Pure Chemical Industries: 169-20365)
1 bottle• Sterile distilled water 20 mL (e.g. Otsuka Pharmaceutical: 36A1X00001)
1 bottles• Sterile cotton buds (e.g. JAPAN COTTON BUDS: 10A754D)
1 box

2.4

OTHERS

2.4.1

EQUIPMENT / INSTRUMENTS
•
•
•
•
•
•
•
•
•
•
•
•
•
•

2.4.2
•
•
•
•

Safety cabinet (or clean bench)
Water bath (37 °C)
CO2 incubator (37 °C, 5% CO2, capable of maintaining high humidity)
Autoclave
96-well multi-plate reader (required filters: 570 nm, 650 nm)
Precision balance (0.1 mg)
Aspirator
Stop-watches
Adjustable micro-pipette (10-200 μL, 200-1000 μL)
Positive-displacement pipette (50 μL)
Sharp-edged forceps (sterile)
Micro spatula (sterile)
Beaker (1-2 L: sterile)
Sterilizable poly wash bottle (500-1000 mL: sterile)

CONSUMMABLE ITEMS
Micro-pipette tips (sterile: 10-200 μL, 200-1000 μL)
Capillary Pistons for positive-displacement pipette (50 μL)
Microtubes (1.5 mL)
Scalpel (KEISEI MEDICAL INDUSTRIAL: Keisei Scalpel 11A)

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3. TEST METHOD*Perform operations in Section 3.1.1-3.1.4 and Section 3.3.1-3.3.2 aseptically in a safetycabinet (or clean bench).
*Operations other than above do not need to be performed with an aseptic technique. For
these operations, refer to Section 2.1.3 INSTRUCTIONS FOR USE OF LabCyte
EPI-MODEL24

3.1

PREPARATIONS

3.1.1

NEGATIVE CONTROL SUBSTANCE
Distilled water is used as negative control.

3.1.2

POSITIVE CONTROL SUBSTANCE
8 mol/L KOH is used as positive control.

3.1.3
POLY WASH BOTTLE FOR PBS(1) Sterilize poly wash bottle using an autoclave.
(2) Fill the sterilized poly wash bottle aseptically with sterile PBS.

3.1.4
MTT SOLUTION(1) Dissolve MTT in the assay medium to prepare the MTT medium (final concentration: 0.5
mg/mL)
Use ultrasonic cleaning equipment or a vortex mixer as necessary in order to completely
dissolve the MTT.
*Store in a dark, cold place and use it within 24 hours.

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3.2
TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT
There are two kinds of test chemicals that interfere with the MTT assay as follows.
(a) Chemicals that stain epidermal tissues.
(b) Chemicals able to directly reduce MTT.
Test chemicals that stain the epidermal tissues have the possibility to transfer from the tissues tothe extraction solution, and to affect the optical density (OD) measurements.
Test chemicals able to directly reduce MTT can affect the optical density (OD) measurements, if
the test chemical is present in the epidermal tissues when the MTT viability test is performed.
Detection procedure of these test chemicals is described below.

3.2.1
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE
3.2.1.1 STEP 1 (PRELIMINARY TEST)(1) Add 50 μL (liquid) or 50 mg (solid) of the test chemical into wells of 24-well assay plate
pre-filled with 0.5 mL of distilled water. Untreated distilled water is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in a CO2 incubator for 60
minutes.
(3) After incubation, shake the mixture gently and visually evaluate the staining of the distilledwater.
(4) If the color of the solution changes significantly, the test chemical is presumed to have thepotential to stain the tissue and a functional check on viable tissues (step 2) should be
performed. If the color of the solution does not change significantly, the test chemical isdetermined not to have the potential to stain the tissue.
(5) Record details of steps (1)-(4) above in the MDS 1-2.
3.2.1.2 STEP 2 (MEASUREMENT OF TISSUE STAINING FOR OD CORRECTION)(1) Add 50 μL (liquid) or 50 mg (solid) of the test chemical, which clearly changed the color ofthe distilled water in step 1, onto the surface of the epidermis tissues. Set a negative control
using distilled water.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST.
However, incubate the tissue for 3 hours in culture media without MTT instead of incubating
in media containing MTT to evaluate the staining of the epidermis tissues.
(3) The corrected OD is calculated using the following formula.
Corrected OD = A − (B − C),where:
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A is the OD of tissue exposed to a test chemical using MTT medium;B is the mean OD of tissue exposed to the test chemical using assay medium instead of
MTT medium; andC is the mean OD of tissue exposed to the negative control using assay medium instead of
MTT medium.
If the OD of (B-C) is below 0, the OD of (B-C) is considered to be 0.
If a corrected OD is below 0, the OD is considered to be 0.

3.2.2
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT
3.2.2.1 STEP 3 (PRELIMINAY TEST)(1) Add 50 μL (liquid) or 50 mg (solid) of the test chemical into wells of 24-well assay plate
pre-filled with 0.5 mL of MTT medium. Untreated MTT medium is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 60
minutes.
(3) After incubation, shake the mixture gently and visually evaluate the staining of the MTT
medium.
(4) If the MTT medium turns blue/purple significantly, the test chemical can reduce MTT and
additional functional check (step 4) must be performed.
(5) Record details of steps (1)-(4) above in the MDS 1-3.

5% SLS

1-bromo hexane

1,1,1-tetrachloroe

5-(1,1-dimethylet

thanehyl)-2-methyl

cinnamaldehyde

eugenol

Photo 1 – Example of test for direct MTT reduction ability (step 3). Test substances5-(1,1-dimethylethyl)-2-methyl, cinnamaldehyde and eugenol have directly reduced
MTT. In these cases, step 4 must be performed.
3.2.2.2 STEP 4 (MEASUREMENT OF DIRECT REDUCE MTT FOR OD CORRECTION)(1) Add 50 μL (liquid) or 50 mg (solid) of the test chemical, which clearly changed the color of
the MTT medium into blue/purple (step 3), onto the surface of the epidermal tissues. Set a
negative control using distilled water.
(2) Follow all procedures described in the SOP Section 3.3 EXECUTION OF THE TEST.
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Instead of using viable epidermal tissues, however, use freeze-killing epidermal tissues.
Freeze-killing tissues were prepared that tissues were frozen at -80°C or lower for 1 hour
and warm-up at 37°C for 30 minutes, and repeat their freeze-thawing step twice. Record the
preparation details of freeze-killing the tissue in the MDS 1-4.
(3) The corrected OD is calculated using the following formula.
Corrected OD = A − (B − C),
Where:A is the OD of viable tissue exposed to a test chemical;
B is the mean OD of freeze-killed tissue exposed to a test chemical; andC is the mean OD of freeze-killed tissue exposed to the negative control.
If the OD of (B-C) is below 0, the OD of (B-C) is considered to be 0.
If a corrected OD is below 0, the OD is considered to be 0.

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EXECUTION OF THE TEST

3.3.1
PREPARATION OF LabCyte EPI-MODEL24 (DAY -1)(1) Pre-warm the assay medium to 37 °C for 30 Figure 1
Assay Plate
minutes using a water bath.
(2) Fill 12 wells of the 1st row and the 3rd row of
1 row(substance application)
each 24-well assay plate with the pre-warmed
2 row
assay medium (0.5 mL/well).
3 row→ Figure 1(holding well)(3) Open the LabCyte EPI-MODEL24 aluminum
4 row(MTT assay)
package.
(4) Open the LabCyte EPI-MODEL24 plate lid
and pick up the culture inserts using sterile forceps.
*Do not touch the epidermis model surface.
*Use forceps to remove agar medium sticking to the outside of the culture inserts.
(5) Transfer the culture inserts into assay
Figure 2
medium-filled wells of the 1st row of the plate
Assay Plateusing sterile forceps.
1 row→ Figure 2(substance application)*Avoid trapping air bubbles under the tissue 2 row
inserts.
3 row(holding well)(6) Place the plate (lid on) in a CO2 incubator.
row(7) Incubate overnight (15-30 hours) until Section 4(MTTassay)
3.2.2 “APPLICATION OF TEST CHEMICALSAND RINSING.”(8) Record details of steps (1) - (7) above in the MDS 2.
st

nd

rd

th

st

nd

rd

th

3.3.2

APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0)

3.3.2.1 PREPARATION OF WELLS FOR MTT ASSAY (4TH ROW)
(1) Pre-warm the MTT medium to 37 °C for 30 minutes using a water bath.
(2) Remove the assay plate from the CO2
Figure 3
incubator.
(3) Open the lid of the assay plate, and fill thewells of the 4th row with the pre-warmed MTT
medium (0.5 mL/well) using a micropipette.
→ Figure 3(4) Record details of steps (1) – (3) above in the
MDS 3.
3.3.2.2

Assay Plate

1st row(substance application)
2nd ro3rd row(holding well)
4th row(MTT assay)

APPLICATION OF TEST CHEMICALS(1) Remove the assay plate from the CO2 incubator.
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(2) Apply test chemicals onto the surface of epidermis tissues in the 1st row of the assay plate.
Use 2 wells per test chemical (N=2).
FOR LIQUIDs: Carefully apply 50 μL of the test chemical onto the center of each epidermis
using a micropipette. After application, close the lid and tap the side of the plate so the liquidspread over the entire epidermis surface. If necessary, use a micro spatula to coat theunapplied tissue surface with the chemical. Do not apply pressure to the epidermis surface.
*Use a positive displacement pipette and tip for viscous liquids. Use the pipette to familiarize
yourself with the nature of the test chemicals in

Photo 2 – Applying a solid substanceadvance.
FOR SOLIDs: Weigh out 50 mg (±2 mg) of the solid
chemical with a precision balance in advance. If
necessary, crush and grind the solid test chemicals
in a mortar with pestle. Apply first 50 μL of distilledwater and then the weighed test chemical onto theepidermis

surface.

Use

a

micro

spatulaifnecessary to gently spread the test chemical.
→ Photo 2*One 24-well assay plate should be used to assay three test chemicals.
→ Figure 4(3 samples x 2(n) = 6(culture inserts))(3) Apply test chemical onto each well at 1-minute intervals.
(4) Incubate each well as described below;

Figure 4

Assay Plate

3 minutes exposure: Incubate each well for 3
minutes in the safety cabinet (lid on between the

1st row

intervals).

2nd row(substance application)

Substance 1

Substance 2

Substance 3(MTT assay)*Close the lid of the assay plate at all times

3rd row

except when applying samples. Leaving the lid

4th row(substance application)

opened might affect the amount of test sample(MTT assay)

due to air circulation in the safety cabinet (orclean bench).
60 minutes exposure: Place the 24-well plates into the incubator (37°C, 5% CO2) for the rest of
the exposure time until 60 minutes is reached for first tissue dosed.
(5) Record details of steps (1)-(4) above in the MDS 3.
3.3.2.3 REMOVAL OF THE TEST CHEMICALS(1) After chemical exposure, open the assay plate and pick up a culture insert with sterile15Version 1.6

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Photo 3 - Rinse 1(2) Discard the test chemical on the tissue by tilting theinsert and tapping it on a beaker. Fill the culture
insert by overflowing it with PBS applied from a poly
wash bottle.
*Apply a stream of PBS directly to the tissue
surface to wash away the test chemical. Maintain auniformly strong stream of PBS while washing thetissue.
→ Photo 3

Photo 4 - Rinse 2*To avoid damaging the tissue with too forceful astream, use a wide-mouth nozzle on the poly
wash bottle.
(3) Tilt the insert to discard the PBS into the beaker.
Remove as much of the PBS inside the culture
insert as possible by tapping it on the beaker.
→ Photo 4(4) Repeat steps (2) and (3) 10 times or more to
remove any residual test chemical as much as possible from the tissue surface.
(5) Using a sterile cotton bud, gently remove any leftover PBS as much as possible from both
inside and outside the culture insert.

Photo 5 - Rinse 3→ Photo 5*Take care to not apply pressure on the surface ofthe tissue with the sterile cotton bud.
(6) If test material remains on the epidermis surface,gently remove it using a sterile cotton bud andrepeat steps (2)-(5).
*If it proves difficult to remove completely all theresidual test chemical from the epidermal tissue
surface, remove as much as possible andcontinue to step 7.
(7) Transfer the blotted culture insert to a well in
the 3

rd

row of the corresponding column(holding well).
→ Figure 5*Avoid trapping air bubbles under the culture

Figure 5

Assay Plate

1st row(substance application)
2nd row

3rd row(holding well)

Substance 1

Substance 2

Substance 3

4th row(MTT assay)

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inserts.
(8) Repeat steps (1)-(7) for all the culture inserts at 1-3-minute intervals in order to keep
exposure periods of each test chemical.
(9) Record details of steps (1)-(8) above in the MDS 3.

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MTT ASSAY (DAY 0)(1) Open the lid of the assay plate, transfer theblotted culture insert to a well in the 4th row of
the corresponding column (for MTT assay).
→ Figure 6(2) Close the lid of the assay plate and place it in
the CO2 incubator.
(3) Incubate for 3 hours.

Figure 6

Assay Plate

1st row(substance application)
2nd row

3rd row(holding well)
4th row(MTT assay)

Substance 1

Substance 2

Substance 3(4) Record details of steps (1)-(3) above in the
MDS 4.

3.3.4

FORMAZAN EXTRACTION AND MEASUREMENT (DAY 0-1)

3.3.4.1

FORMAZAN EXTRACTION(1) Remove the assay plate(s) from the CO2 incubator 3 hours (±5 minutes) after the MTT
assay.

Photo 6 – scratch up the epidermis(2) Open the lid of the assay plate and pick-up thetissue from each culture insert by the pinching itwith forceps (Photo 6) or by cutting both tissue
and membrane filter off from the culture insert witha scalpel.
(3) Transfer the epidermis tissue into a 1.5mL microtube.
(4) Add 300 μL of isopropanol to the micro tubes andimmerse the entire epidermis tissue in the isopropanol.
(5) Incubate the micro tubes in a dark cold place overnight (more than 15 hours) in order tocompletely extract pigments.
*Tighten the micro tube seal.
*Periodically shaking the micro tubes will contribute to a more efficient extraction.
(6) Shake the micro tubes to mix the solution.
*If epidermis tissue fragments are suspended, wait until they sink or gently centrifuge them
(if a centrifuge is available).
(7) Transfer 200 μL of the solution in each micro tube into each well on a 96-well plate.
*One well of 200 μL of isopropanol should be set as a blank.
*Figs. 7A and 7B show typical sample allocation on a 96-well plate for both for non-corrected
tissues and for corrected tissues.
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Figure 7A – Sample allocation for a 96-well plate(same pattern for 3 or 60 minutes application)
1

2

A

Blank

B

S1-1
S6-1
S11-1
S16-1
S21-1
S26-1

C
D
E
F
G

3

S1-2
S6-2
S11-2
S16-2
S21-2
S26-2

4

5

DW-1

DW-2

S2-1
S7-1
S12-1
S17-1
S22-1
S27-1

S2-2
S7-2
S12-2
S17-2
S22-2
S27-2

6

7

S3-1
S8-1
S13-1
S18-1
S23-1
S28-1

S3-2
S8-2
S13-2
S18-2
S23-2
S28-2

8

KOH-1

9

[Only 60 min] [Only 60 min]

S4-1
S9-1
S14-1
S19-1
S24-1
S29-1

10

11

S5-1
S10-1
S15-1
S20-1
S25-1
S30-1

S5-2
S10-2
S15-2
S20-2
S25-2
S30-2

KOH-2
S4-2
S9-2
S14-2
S19-2
S24-2
S29-2

12

H

Figure 7B – Sample allocation for a 96-well plate for OD-correcting tissue(same pattern for 3 or 60 minutes application)
1
A
B
C
D
E
F
G

2

Blank
S1-1
S6-1
S11-1
S16-1
S21-1
S26-1

3

S1-2
S6-2
S11-2
S16-2
S21-2
S26-2

4

DW-1
S2-1
S7-1
S12-1
S17-1
S22-1
S27-1

5

DW-2
S2-2
S7-2
S12-2
S17-2
S22-2
S27-2

6

7

8

9

10

11

S3-1
S8-1
S13-1
S18-1
S23-1
S28-1

S3-2
S8-2
S13-2
S18-2
S23-2
S28-2

S4-1
S9-1
S14-1
S19-1
S24-1
S29-1

S4-2
S9-2
S14-2
S19-2
S24-2
S29-2

S5-1
S10-1
S15-1
S20-1
S25-1
S30-1

S5-2
S10-2
S15-2
S20-2
S25-2
S30-2

12

H

(8) Record details of steps (1)-(6) above in the MDS 5.
3.3.4.2

OPTICAL DENSITY MEASUREMENTS OF THE EXTRACTS(1) Using a 96-well plate reader, measure optical densities (OD) at 570 nm and 650 nm anddetermine the measured OD by subtracting the 570 nm OD from the 650 nm OD.
The equation is shown below:
Measured OD = [570 nm ODsample – 570 nm ODblank] – [650 nm ODsample – 650 nm ODblank]*Set the plate reader-calculated value as the measured OD if the 96-well plate reader performs
automatic calculations.

(2) Calculate the cell viability of a sample using the equation below. Furthermore, calculate thedifference of viability.
(3) Record details of steps (1) and (2) above in the MDS 5.

Cell Viability (%)＝

Measured ODsample
Mean Measured ODNC

×100

19

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4. ASSESSMENT
4.1

CONDITIONS FOR A SUCCESSFUL STUDY

The skin corrosion test should be considered successful if the following criteria have been met.
•

Cell viability: 0.7 ≤ mean OD (A570/650) measured value for negative control ≤ 2.5.

•

Positive control (60 minutes exposure): mean cell viability for 8N KOH < 15%.

•

In the viability range of 20-100%, and ODs ≥0.3, the difference of viability between the twotissue replicates should not exceed 30%.

4.2

ASSAY CRITERIACorrosive potential of the test materials is predicted from the relative mean tissue viabilities
obtained after 3 minutes, as well as 60 minutes treatment, compared to the negative control
tissues concurrently treated with H2O. The prediction models for the LabCyte EPI-MODEL24 SCT
methods, associated with the UN GHS classification system, are shown in Table 3.
Table 3. LabCyte EPI-MODEL24 SCT prediction model
Step 13 minutes
60 minutes
Prediction< 50%
Corrosive
Viability
< 15%
≥ 50%
≥ 15%
Non-corrosive

Step 23 minutes
< 15%
≥ 15%

Prediction
Sub-category 1A
Sub-category 1Band 1C20

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REFFERENCE(1) OECD (2001) Harmonised Integrated Classification System for Human Health andEnvironmental Hazards of Chemical Substances and Mixtures. OECD Series on Testingand Assessment Number 33. ENV/JM/MONO(2001)6, Paris.
http://www.olis.oecd.org/olis/2001doc.nsf/LinkTo/env-jm-mono(2001)6(2) Fentem, J.H., et al. (1998). The ECVAM international validation study on in vitro tests forskin corrosivity. 2. Results and evaluation by the Management Team. Toxicol. in Vitro 12,483-524.
(3) Liebsch, M., et al. (2000). The ECVAM prevalidation study on the use of EpiDerm for skincorrosivity testing. ATLA 28, 371-401.
(4) OECD (2016) OECD Guideline for the testing of chemicals, No.431: In vitro skin corrosion:reconstructed human epidermis (RHE) test method. Paris, France: Organization foreconomic cooperation and development.

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MDS 1-1:
RECEIPT OF LABCYTE EPI-MODEL24
Laboratory name:1.

Test name:

Test No. :LabCyte EPI-MODEL24
Date received：
Lot No.：Expiration date：(MM/DD/YYYY)

Accessories： Assay medium, 30mL □（Lot No.：

24 well assay plate □Expiration date：）(MM/DD/YYYY)

Note

2.

Assay medium
Date received：
Lot No.：Expiration date：(MM/DD/YYYY)

Note

Date:

(MM/DD/YYYY)

Secretariat

Operator:
Check date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Study director:

Name:

22

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MDS 1-2:
MTT REACTION INTERFERENCE TEST (DETECTION OF THE CHEMICALS THAT STAIN
THE TISSUE)
STEP-1 (Section 3.2.1.1)
Laboratory name:1.

Test name:

Test no. :Add distilled water (0.5 mL) to the wells of the 24-well assay plate.
To add distilled water (0.5 mL)□

Execution date/time:2.

Apply test chemicals to the wells of the 24-well assay plate.

3.

Incubate the 24-well assay plate in a CO2 incubator for 60 minutes.
Incubation starting time:
Incubation ending time:

4.

Check the color of water.

5.

Test chemical information and check list of coloring potential.
Test chemical

Physical state

AmountH2O (NC)

Liquid50μL

Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid

50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (

Coloring potential

mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)

Note

Date:
Secretariat

Operator:
Check date:

Check date:Study director:

Name:

23

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MDS 1-3:
MTT REACTION INTERFERENCE TEST (DETECTION OF CHEMICALS THAT DIRECTLY
REDUCE MTT)
STEP-3 (Section 3.2.2.1)
Laboratory name:1.

2.

Test name:Preparation of MTT medium

Preparation vol.

mL

Test no. :Lot No.

Execution date/time:(MM/DD/YYYY HH/MM)

Add MTT medium (0.5 mL) to the wells of the 24-well assay plate.
To add MTT medium (0.5 mL)□

Execution date/time:3.

Apply test chemicals to the wells of the 24-well assay plate.

4.

Culture the 24-well assay plate in CO2 incubator for 60 minutes.

MTT reaction starting time:
MTT reaction ending time:

5.

Check the color of MTT medium.

6.

Test chemical information and checked list of MTT reducing potential.

Test chemical

Physical state

AmountH2O (NC)

Liquid50μL

Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid
Liquid, viscous, solid

50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (
50μL, (

Coloring potential

mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)
mg)

Note

Date:
Secretariat

Operator:
Check date:

Check date:Study director:

Name:

24

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MDS 1-4:
PREPARATION OF FREEZE-KILLED TISSUE (Section 3.2.2.2)
Laboratory name:

Test name:

Test no. :1.

Transfer LabCyte EPI-MODEL24 tissues to a 50 mL tube or appropriate sterile container.

2.

Freeze tissues in a -80℃ deep-freezer for 1 hour (1 freezing).

stStore for 1 hour.

3.

□

Date/time:Thaw tissues in a 37℃ incubator for 30 minutes.
Store for 30 minutes.

4.

□Date/time:

Freeze tissues in a -80℃ deep-freezer for more than 1 hour (2Store for 1 hour.

5.

□

nd

freezing).

Date/time:Thaw tissues in a 37℃ incubator for 30 minutes.
Store for 30 minutes.

□

Date/time:

Note

Date:
Secretariat

Operator:
Check date:

Check date:Study director:

Name:

25

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MDS 2:
PRE-INCUBATION OF LABCYTE EPI-MODEL24 (Section 3.3.1)
Laboratory name:

Test name:

Test No. :
st

1.

rd

Warm up the assay medium and add 0.5 mL of the assay medium to the wells of the 1 and 3
row on the 24-well assay plate.
Assay medium：（Lot No.：

Expiration date：）(MM/DD/YYYY)

Warm for 30 minutes.

□

Add 0.5 mL of assay medium to each well.
Number of plates：

□

st

2.

Transfer culture inserts to wells in the 1 row on the 24-well assay plate.
LabCyte EPI-MODEL24：（Lot No.：

Expiration date：）(MM/DD/YYYY)

Execution date/time：(MM/DD/YYYY HH:MM)

Release any air bubbles trapped under the cell culture insert.

3.

□

LabCyte EPI-MODEL24 is cultured in a CO2 incubator overnight.

.

Culture starting date/time：(MM/DD/YYYY HH:MM)

Planned date/time of chemical exposure：(MM/DD/YYYY HH:MM)

Note

Date:

(MM/DD/YYYY)

Secretariat

Operator:
Check date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Study director:

Name:

26

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MDS 3-1:
APPLICATION OF TEST CHEMICALS AND RINSING: 3 MINUTES EXPOSURE(Section 3.3.2)
Laboratory name:1.

2.

Test name:Preparation of MTT medium

Preparation vol.

mL

Lot No.

Execution date/time：(MM/DD/YYYY HH/MM)thWarm up the MTT medium and add 0.5mL to the wells in the 4 row on the 24-well assay plate.
MTT medium.：（Lot No.：
Warm for 30 minutes □3.

Expiration date：

Add 0.5 mL of the MTT medium □Execution date/time：(MM/DD/YYYY HH/MM)

Apply test chemicals to the LabCyte EPI-MODEL24.
Ending date/time：(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)

Test chemical information

Test chemical code No.

Lot No.

Distilled Water(Negative control)

5.

）(MM/DD/YYYY)

Starting date/time：

4.

Test No.:

Physical state

Test chemical vol.(weight)(measured weight)

Time ofapplication

Exposure time
（3 minutes.）

Liquid50μL：□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

Liquid, viscous, solid

50μL, (

mg,

mg)

：

□

After exposure to test chemical for 3 minutes, wash out the LabCyte EPI-MODEL24 and transferrdthe culture inserts to the 3 row on the 24-well assay plate for holding wells.
PBS：
Starting date/time：(MM/DD/YYYY HH/MM)（Lot No.：

Expiration date：）(MM/DD/YYYY)

Ending date/time：(MM/DD/YYYY HH/MM)

Release any air bubbles trapped under the cell culture insert.

□

27

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Note

Date:

(MM/DD/YYYY)

Secretariat

Operator:
Check date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Study director:

Name:

28

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MDS 3-2:
APPLICATION OF TEST CHEMICALS AND RINSING: 60 MINUTES EXPOSURE(Section 3.3.2)
Laboratory name:1.

2.

Test name:Preparation of MTT medium

Preparation vol.

mL

Lot No.

Execution date/time：(MM/DD/YYYY HH/MM)thWarm up the MTT medium and add 0.5mL to the wells in the 4 row on the 24-well assay plate.
MTT medium.：（Lot No.：

Expiration date：

Add 0.5 mL of the MTT medium □Execution date/time：(MM/DD/YYYY HH/MM)

Apply test chemicals to the LabCyte EPI-MODEL24.
Starting date/time：

Ending date/time：(MM/DD/YYYY HH/MM)

4.

）(MM/DD/YYYY)

Warm for 30 minutes □3.

Test No.:(MM/DD/YYYY HH/MM)

Culture LabCyte EPI-MODEL 24 in a CO2 incubator for 60 minutes.

Culture starting date/time：(MM/DD/YYYY HH:MM)

Culture ending date/time：(MM/DD/YYYY HH:MM)

5.

Test chemical information
Test chemical code No.

Lot No.

Distilled Water (Negative
control)
8N KOH(Positive control)

6.

Physical state

Test chemical vol.(weight)(measured weight)

Time ofapplication

Exposuretime(60 minutes）

Liquid50μL：□

LiquidLiquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid
Liquid, viscous,
solid

50μL

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

50μL, (

mg,

mg)

：

□

After exposure to test chemical for 60 minutes, wash out the LabCyte EPI-MODEL24 and transferrdthe culture inserts to the 3 row on the 24-well assay plate.
PBS：
Starting date/time：(MM/DD/YYYY HH/MM)（Lot No.：

Expiration date：）(MM/DD/YYYY)

Ending date/time：(MM/DD/YYYY HH/MM)

Released any air bubbles trapped under the cell culture insert.

□

29

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Note

Date:

(MM/DD/YYYY)

Secretariat

Operator:
Check date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Study director:

Name:

30

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MDS 4:MTT ASSAY (Section 3.3.3)
Laboratory name:

Test name:

Test No. :th

1.

After washing, transfer the LabCyte EPI-MODEL24 to wells of the 4 row of 24-well assay plate.
3 minutes exposure:Starting date/time：

Ending date/time：(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)

60 minutes exposure:Starting date/time：

Ending date/time：(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)

Release any air bubbles trapped under the cell culture insert.
2.

□

Incubate LabCyte EPI-MODEL24 in a CO2 incubator for 3 hours.
3 minutes exposure:
MTT reaction starting date/time：(MM/DD/YYYY HH/MM)

60 minutes exposure:
MTT reaction starting date/time：(MM/DD/YYYY HH/MM)

MTT reaction ending date/time：(MM/DD/YYYY HH/MM)

MTT reaction ending date/time：(MM/DD/YYYY HH/MM)

Note

Date:

(MM/DD/YYYY)

Secretariat

Operator:
Check date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Study director:

Name:

31

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May, 2017

Japan Tissue Engineering Co., Ltd.

MDS 5:FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.4)
Laboratory name:1.

Test name:

Test No. :After MTT reaction, use forceps to pick up the cultured epidermis from the cell culture insert andput it in a 1.5 mL microtube.
Did you use a scalpel to cut out the cultured epidermis?
Execution date：□(MM/DD/YYYY)

2.

Add isopropanol (300μL) to a microtube and completely immerse the tissue in isopropanol.
Isopropanol
Lot No.
Immersion of the cultured epidermis in isopropanol.
Execution date：

Added isopropanol (300μL)□□(MM/DD/YYYY)

3.

For MTT formazan extraction, leave the micro tubes in a cold and dark space for more than 15hours.

Place micro tube in a cold and dark space.

4.

□

Transfer the extracted solution (200μL) to a well of a 96-well plate.
Transfer to the 96-well plate.
Execution date/time：□(MM/DD/YYYY HH/MM)

Sample location on 96-well plate.
1

2

A

Blank

B

S1-1

C

3

4

5

6

7

8

KOH-1

9

10

11

KOH-2

12

DW-1

DW-2

S1-2

S2-1

S2-2

S3-1

S3-2

S4-1

S4-2

S5-1

S5-2

S6-1

S6-2

S7-1

S7-2

S8-1

S8-2

S9-1

S9-2

S10-1

S10-2

D

S11-1

S11-2

S12-1

S12-2

S13-1

S13-2

S14-1

S14-2

S15-1

S15-2

E

S16-1

S16-2

S17-1

S17-2

S18-1

S18-2

S19-1

S19-2

S20-1

S20-2

F

S21-1

S21-2

S22-1

S22-2

S23-1

S23-2

S24-1

S24-2

S25-1

S25-2

G

S26-1

S26-2

S27-1

S27-2

S28-1

S28-2

S29-1

S29-2

S30-1

S30-2

[Only 60min]

[Only 60
min]

H

5.

Analyze extract OD at 570 nm and 650 nm, and calculate the OD (570 nm-650 nm).

Analyze OD at 570 nm and 650 nm.
Calculate the OD (570 nm-650 nm).
Calculate cell viability and difference of viability.
Cell viability and difference of viability are recorded on a separate data sheet.
The data sheet is attached to the back of this sheet.
Check for input errors.
Execution date/time：

□
□
□
□
□
□

(MM/DD/YYYY HH/MM)

Note
Date:

(MM/DD/YYYY)

Secretariat

Operator:
Check date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Study director:

Name:

32

Version 1.6

IN VITRO SKIN CORROSION TEST:HUMAN EPIDERMIS MODEL
Model: LabCyte EPI-MODEL24

May, 2017

Page 33 of 33

Japan Tissue Engineering Co., Ltd.

REVISION HISTORY
Rev.

Content

Date Revised

Ver. 1.1

First version

01/05/2015

Ver. 1.2

Revised clerical error.

02/03/2017

Ver. 1.3

Replaced the term “tissue viability” by “cell viability” throughout entire

02/06/2017

document.
In the section 3.3. “EXECUTION OF THE TEST”, additionally explainedthat the solid test chemical is crush and grind in a mortar with pestle ifnecessary.
Ver. 1.4

Revised clerical error.

03/03/2017

In the section 3.2. “TEST FOR DETECTING CHEMICALS THATINTERFERE WITH MTT ENDPOINT”, additionally explained the
preparation of freeze-killing tissues.
Ver. 1.5

In the section 3.2. “TEST FOR DETECTING CHEMICALS THAT

03/23/2017

INTERFERE WITH MTT ENDPOINT”, supplementary explained
calculating formula for correcting OD.
Ver. 1.6

Revised clerical error.

05/12/2017

In the section 3.2. “TEST FOR DETECTING CHEMICALS THATINTERFERE WITH MTT ENDPOINT”, added directions concerning thenegative control in STEP 2 and STEP 4.
In the section 3.3.3. “REMOVAL OF THE TEST CHEMICALS”, added
detailed explanation of the washing procedure.
Inthesection

3.3.4.

“FORMAZANEXTRACTIONAND

MEASUREMENT”, added description of two different methods toremove tissues from the culture inserts.

33